Proteins expressed in eukaryotic expression systems undergo a process of post-translational modification, which involves glycosylation. Eukaryotic expression systems which have been established today for the production of glycoproteins, like IgG and other monoclonal antibodies comprising an Fc region add N-glycans to the polypeptide chains.
In IgG, the most important N-glycan is bound at Asn 297 of both CH2 chains (see FIG. 14), which comprises, among others, N-acetyl-neuraminic acid (sialic acid), N-acetyl-glucosamine, galactose, mannose, and fucose residues.
This applies, basically, for transgenic plant expression systems as well as for mammalian cell lines, insect cell lines etc. In all these cases, the N-glycan comprises at least one fucose residue which is bound either α-3-glycosidically or α-6-glycosidically to the N-acetyl-glucosamine residue bound to the Asn residue of the polypeptide chain.
Yeast expression systems tend to produce hyperglycoproteins rich in mannose, which often lead to unwanted immune reactions when the therapeutic antibody is administered to a patient. Baculovirus transfected insect cell systems cause problems due to hypoglycosylation, which negatively affects the effector function of therapeutic antibodies. Furthermore, the major disadvantage are the catalytic properties of infectious baculovirus that narrows the window for full IgG production.
ADCC is a mechanism of cell-mediated immunity whereby an effector cell of the immune system actively lyses a target cell that has been bound by specific antibodies. It is one of the mechanisms through which antibodies, as part of the humoral immune response, can act to limit and contain infection. Classical ADCC-mediating effector cells are natural killer (NK) cells; but monocytes and eosinophils can also mediate ADCC. ADCC is part of the adaptive immune response due to its dependence on a prior antibody response.
Therapeutic antibodies which are used to elicit an ADCC in target cells need an Fc region in order to be recognized by Fc gamma receptors of the said effector cells.
Recent studies have shown that monoclonal antibodies having a reduced amount of fucose in its glycosylation pattern exhibit much higher Antibody-Dependent Cellular Cytotoxicity (ADCC) activity as compared to fucosylated antibodies. Again, it is basically position Asn 297 where a lack of fucose residues leads to the increased ADCC. The mechanism behind the increased ADCC of a low/no-fucose Antibody seems to be mediated by an increased affinity of a so modified Fc region to FcγR, for example FcγIIIa (CD 16), the major Fc receptor for ADCC in human immune effector cells (Shields et al, 2002).
Fucosylation is one of the most common modifications involving oligosaccharides on glycoproteins or glycolipids. Fucosylation comprises the attachment of a fucose residue to N-glycans, O-glycans, and glycolipids. O-Fucosylation, a special type of fucosylation, is very important for Notch signaling. The regulatory mechanisms for fucosylation are complicated. Many kinds of fucosyltransferases, the GDP-fucose synthesis pathway, and GDP-fucose transporter are involved in the regulation of fucosylation.
Glycosylation is known to impact the effector functions of therapeutic monoclonal antibodies. Among the various sugar residues in the oligosaccharide chain of an antibody, fucose is one of the key sugars that affects the antibody dependent cellular cytotoxicity (ADCC) induced by the product.
Manipulation of cell culture parameters is often employed to control galactosylation and sialylation of an antibody. Control of fucosylation is majorly done by using FUT8 knock out cells and other gene silencing models through cell line engineering.
US20090208500 discloses the production of antibodies with reduced fucose and improved Fc function by manipulation of FUT8 Knock out cells.
U.S. Pat. No. 7,972,810 discloses cell culturing methods and media containing manganese that improve glycosylation or sialylation of glycoproteins, including erythropoietin and analogs or derivatives thereof. According to the disclosure, manganese increases sialylation and site occupancy in case of O-linked and N-linked glycosylation (i.e. lower aglycosylated product) and also increases terminal galactosylation.
Further, fucose content of monoclonal antibodies can be controlled by culture medium osmolality for high antibody-dependent cellular cytotoxicity. (Konno et al. 2012)
Yet, there is a need for an efficient method of producing glycoproteins in a desired cell line while controlling the fucose content of the recombinantly engineered antibodies without undergoing the laborious process of creating a FUT8 gene knockout in a selected cell line each time.